Retinopathy is one of the most debilitating complications of diabetes, but the mechanisms that lead to its development are poorly understood. In the pathogenesis of diabetic retinopathy, retinal capillary cells undergo apoptosis, which precedes the development of histopathology. The proposed studies are aimed at understanding the putative regulatory role(s) of Ras, a small molecular weight GTP-binding protein, in retinal capillary cell death in diabetes. In support of this, we have provided exciting preliminary data showing that the protein expression and mRNA levels of Ras are increased in the retina in diabetes and in retinal endothelial cells cultured in high glucose medium, and specific inhibitors of Ras function also inhibit glucose-induced capillary cell death. The central hypothesis of this proposal is that Ras activation plays a crucial role in retinal cell death, and ultimately the development of diabetic retinopathy. The first specific aim will determine the mechanism by which Ras is activated in retinal capillary cells in diabetes. The mechanism(s) by which glucose activates Ras will be determined by measuring its expression, and increase in GTP loading onto Ras in isolated retinal capillary cells in culture and in the retina obtained from diabetic rats. The role of Ras activation in glucose-induced increase in retinal capillary cell death will be determined by using specific inhibitors of Ras function, and via the transfection approaches involving the dominant negative Ras mutant. Activation of Ras in the retina in diabetes will be compared with the capillary cell death, and temporal relationship to the development of histopathology will be established. Since Ras activation has been shown to recruit its regulatory protein, Raf-1, and Ras/Raf complexation initiates a signaling cascade, the second specific aim will define the Ras-dependent cellular signaling mechanisms underlying hyperglycemia-induced retinal capillary cell death. Understanding the signal transduction mechanisms involved in retinal capillary cell death in diabetes will provide fresh insight into the development of retinopathy, and will help elucidate novel molecular targets for future pharmacological interventions to halt/retard this sight-threatening complication of diabetes. [unreadable] [unreadable]